MPRAsnakeflow experiment QC report

TipGood Spearman correlation across replicates!

The data has a good Spearman correlations across replicates using a barcode threshold per oligo of 10! Spearman correlation of DNA is 1.00, RNA is 0.98 and DNA/RNA ratio is 0.96.

Overall quality metrics

Table explanation
  • median rna read count: Median of RNA read count for oligos that passed filtering to determine sufficient coverage in terms of read count. Value is the median of all replicates.
  • median barcodes passing filtering: Median number of barcodes across tested sequences that passed filtering to determine if there was sufficient barcode to oligo coverage. Value is the median of all replicates.
  • pearson correlation: The correlation of log2 RNA/DNA ratios across tested sequences as a measure of replicable activity signal. Value is the median of replicate comparisons using only oligos with >= 10 barcodes.
  • fraction oligos passing: Fraction of tested sequences that passed filtering of the mappable sequences to determine if the designed library was sufficiently recovered. Value is the median of all replicates and using only oligos with >= 10 barcodes.
median barcodes passing filtering median rna read count pearson correlation fraction oligos passing
176 12224 0.97 0.96

DNA over RNA counts

Plotting normalized counts of DNA vs RNA (median across replicates). Only oligos within all replicates are shown. We should see a variation within the RNA count data (along the y axis). If count data between RNA and DNA is highly correlated (e.g. follows the identity line) there is no variation between designed oligos. This is an indication that RNA is inflated with DNA and the DNA digestion before creating cDNA did not work as expected.

Oligo correlation

Oligo correlation plots of DNA, RNA and DNA/RNA ratios across replicates. First tab shows plots using (in average) 2302 oligos with a minimum number of 10 barcodes. Second tab shows all 2391 oligos that have assigned barcodes.

Condition A B #Oligos A #Oligos B #Oligos Joined DNA spearman RNA spearman Ratio spearman DNA log2 pearson RNA log2 pearson Ratio log2 pearson
HEPG2 1 2 2303 2302 2302 1.00 0.98 0.96 1.00 0.98 0.96
HEPG2 1 3 2303 2303 2303 1.00 0.98 0.97 1.00 0.98 0.97
HEPG2 2 3 2302 2303 2302 1.00 1.00 0.99 1.00 1.00 0.99
Condition A B #Oligos A #Oligos B #Oligos Joined DNA spearman RNA spearman Ratio spearman DNA log2 pearson RNA log2 pearson Ratio log2 pearson
HEPG2 1 2 2391 2391 2391 0.99 0.98 0.96 0.98 0.98 0.95
HEPG2 1 3 2391 2391 2391 1.00 0.98 0.97 0.99 0.98 0.97
HEPG2 2 3 2391 2391 2391 0.99 0.99 0.99 0.99 0.99 0.98

Experiment statistic

The total number of oligos in this experiment is 2398 (defined by the assignment) with 938503 associated barcodes.

In average across replicates we see 2391 from 458820 average barcodes in the count data and around 377578 barcodes where not in the assignment.

condition replicate oligos dna/rna matched barcodes unknown barcodes dna/rna % matched barcodes total dna counts total rna counts avg dna counts per bc avg rna counts per bc barcode outlier removed avg dna/rna barcodes per oligo
HEPG2 1 2391 458306 376572 54.89 21004963 55170372 25.16 66.08 0 191.68
HEPG2 2 2391 455280 295548 60.64 20013815 36062281 26.66 48.03 0 190.41
HEPG2 3 2391 462874 460614 50.12 29614804 55689525 32.07 60.30 0 193.59
Experiment Barcodes Counts Average counts Assigned barcodes Assigned counts Average assigned counts Fraction assigned barcodes Fraction assigned counts
HEPG2.1.DNA 1712727 21963798 12.82 469743 17029998 36.25 0.27 0.78
HEPG2.2.DNA 1643842 20988683 12.77 467728 16298744 34.85 0.28 0.78
HEPG2.3.DNA 2111923 30931142 14.65 477864 24014862 50.25 0.23 0.78
HEPG2.1.RNA 2779709 57511512 20.69 487105 43586626 89.48 0.18 0.76
HEPG2.2.RNA 2198921 37761918 17.17 482087 28543781 59.21 0.22 0.76
HEPG2.3.RNA 2777366 57875305 20.84 487857 43863504 89.91 0.18 0.76

Histograms barcodes per oligo, counts per barcode

Histogramm of number of barcodes per oligo and the number of counts per barcode devidied by DNA and RNA. Median is red, mean is blue.

Activity

Violin and box plots of the log2 fold change of all oligos by the assay. Grouped by labels if set, otherwise NA. First tab shows plots using (in average) 2302 oligos with a minimum number of 10 barcodes. Second tab shows all 2391 oligos that have assigned barcodes.